Abstract
Therapeutic intervention targeting mRNA typically aims at reducing the levels of disease-causing sequences. Achieving the opposite effect of blocking the destruction of beneficial mRNA remains underexplored. The degradation of mRNA starts with the removal of poly(A) tails reducing their stability and translational activity which is mainly regulated by the CCR4-NOT complex. The subunit NOT9 binds various RNA binding proteins which recruit mRNA in a sequence-specific manner to the CCR4-NOT complex to promote their deadenylation. These RNA binding proteins interact with NOT9 through a helical NOT9 binding motif which we used as a starting point for development of the hydrocarbon stapled peptide NIP-2. The peptide (KD = 60.4 nM) was able to inhibit RNA-binding (IC50 = 333 nM) as well as the deadenylation activity of the CCR4-NOT complex in vitro while being cell-permeable (EC50 = 2.44 μM). A co-crystal structure of NIP-2 bound to NOT9 allowed further optimization of the peptide through point mutation leading to NIP-2-H27A-N3(KD = 122 nM) with high cell permeability (cell-permeability EC50 = 0.34 μM). The optimized peptide was able to inhibit deadenylation of target mRNAs when used in HeLa cells at a concentration of 100 μM demonstrating the feasibility of increasing mRNA stability.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.